Human Papillomaviruses (HPVs) are DNA tumor viruses causing cervical, anogenital and skin cancers. HPV virions consist of a molecule of double-stranded circular DNA contained within an icosahedral capsid composed of L1 major capsid protein and L2 minor capsid protein. Nuclear import of HPV capsid proteins and genome is crucial for papillomavirus replication cycle. E6 and E7 are the transforming proteins of high risk HPVs, and regulation of their nuclear import may play an important role in the transformation process. My laboratory pioneered the study of the nuclear import pathways of HPV proteins and we have identified and characterized these pathways for L1 major and L2 minor capsid proteins as well as for E6 oncoproteins both for high risk HPV16 and low risk HPV11. In addition, we discovered that high risk HPV16 E7 oncoprotein enters the nucleus via a novel Ran-dependent import mechanism. HPV infection depends on the import of the viral genome into the nucleus of infected host cells but how the viral DNA enters the nucleus is unknown. Our hypothesis, based on our previous data on nuclear import of L2 proteins and their interactions with the DNA, is that L2 may facilitate nuclear import of papillomavirus DNA during the initial phase of infection. In Specific Aim 1 we will analyze the role of L2 proteins in nuclear import of papillomavirus DNA, determine the nuclear localization signal (NLS) and DNA binding domain involved and characterize in detail the nuclear import pathways for the BPV1 and HPV16 genomes. In order to accomplish these goals we will use a combination of in vitro nuclear import assays, mutagenesis, binding experiments, and in vivo localization studies. In Specific Aim 2 we will continue to dissect the novel nuclear import mechanism for HPV16 E7 oncoprotein, map its novel NLS and compare the nuclear import of high risk HPV16 E7 versus low risk HPV11 E7. We will also use mutational analysis of high risk HPV16 E7 to obtain E7 mutants that are defective in nuclear import but still able to interact with pRB and other major targets of E7. These E7 mutants will be used to gain insight into the role of E7 nuclear import in binding and targeting pRB for degradation in vivo. Overall, the studies proposed in this renewal application will continue to advance our understanding of major nuclear import pathways relevant to the papillomavirus infection and pathogenesis.